Generation of fully human anti-vascular endothelial growth factor 165 monoclonal antibody with antitumor efficacy from transgenic five-feature mice

Aim ：To prepare a fully human anti-VEGF₁₆₅(vascular endothelial growth factor 165) monoclonal antibody with antitumor activity from five-feature mice which express human immunoglobin loci. Methods ：A routine method for the generation of monoclonal antibodies(mAbs) against the human VEGF₁₆₅ was developed.The immunizing effect between five-feature mice and BALB/c was observed and the mAb was purified through MBP IgM _{affinity chromatography.The effect of mAbs on antitumor was tested in vitro by T24 cell line. Results ：Four hybridomata secreting mAbs steadily were isolated successfully，and the serum titer of mAb in BALB/c mice was almost 10 times higher than that in five-feature mice.The indirect ELISA method for mAb titer determination was also established.The anti-VEGF165mAb was purified to homogeneity by precipitation with ammonium sulfate followed by the affinity chromatography on MBP IgM purification column.Moreover，both purified human IgM V2，V75 and mouse ascites were characterized by SDS-PAGE and Western blotting.Proliferation of T24 cell line was considerably inhibited by V2 and V75. Conclusion ：Five-feature mice could be used to produce fully human monoclonal antibody.The fully human anti-VEGF mAb is potential in the cancer treatment.}